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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/06—Uses of viruses as vector in vitro

Definitions

• the present invention relates to a recombinant receptor, comprising an extracellular ligand-binding domain and a cytoplasmic domain that comprises a heterologous bait polypeptide, which receptor is activated by binding of a ligand to said ligand binding domain and by binding of a prey polypeptide to said heterologous bait peptide.
• the present invention also relates to a method to detect compound-compound-binding using said recombinant receptor.
• Protein-protein interactions are an essential key in all biological processes, from the replication and expression of genes to the morphogenesis of organisms. Protein- protein interactions govern amongst others ligand-receptor interaction and the subsequent signaling pathway; they are important in assembly of enzyme subunits, in the formation of biological supramolecular structures such as ribosomes, filaments and virus particles and in antigen-antibody interactions.
• researchers have developed several approaches in attempts to identify protein-protein interactions. Co-purification of proteins and co-immunoprecipitation were amongst the first techniques used. However, these methods are tedious and do not allow high throughput screening. Moreover, they require lysis corrupting the normal cellular context.
• WO9002809 describes how a binding protein can be displayed on the surface of a genetic package, such as a filamentous phage, whereby the gene encoding the binding protein is packaged inside the phage. Phages, which bear the binding protein that recognizes the target molecule are isolated and amplified.
• Several improvements of the phage display approach have been proposed, as described e.g. in WO9220791 , WO9710330 and WO9732017.
• all these methods suffer from the difficulties that are inherent at the phage display methodology: the proteins need to be exposed at the phage surface and are so exposed to an environment that is not physiological relevant for the in vivo interaction.
• modification- dependent phage display systems have not been described.
• US5637463 describes an improvement of the yeast two-hybrid system, whereby can be screened for modification dependent protein-protein interactions.
• this method relies on the co-expression of the modifying enzyme, which will exert its activity in the cytoplasm and may modify other enzymes than the one involved in the protein-protein interaction, which may on its turn affect the viability of the host organism.
• FIG. 1 A schematic representation of the invention is given in Figure 1. It is one aspect of the present invention to provide a recombinant transmembrane receptor, comprising an extracellular ligand binding domain and a cytoplasmic domain that comprises a heterologous bait polypeptide, which receptor is activated by binding of a ligand to said ligand binding domain and by binding of a prey polypeptide to said heterologous bait polypeptide.
• the recombinant receptor can be a chimeric receptor, in which the ligand binding domain and the cytoplasmic domain are derived from two different receptors.
• the receptor is a multimerizing receptor; this can be a homomultimerizing receptor as well as a heteromultimerizing receptor.
• the cytoplasmic domain of the recombinant receptor comprises a heterologous bait polypeptide, which can be fused to the carboxyterminal end, or can replace a part of this carboxyterminal end or can be situated in the cytoplasmic domain itself, as an insertion or a replacement of an endogenous internal fragment.
• a heteromultimerizing receptor not all the chains need to comprise the bait, but it is sufficient if one of the composing chains does comprise the bait in its cytoplasmic domain.
• At least one of the activation sites in the cytoplasmic domain of the receptor has been inactivated, so that the receptor is not activated and there is no active signaling pathway if only a ligand is binding to the ligand-binding domain of said recombinant receptor.
• Such inactivation can be obtained in several ways, such as by replacement of the amino acid, which can be activated, by another amino acid, by changing the amino acid context of the activation site or by deleting the activation site. Insertion of the heterologous bait polypeptide and inactivation of the activation sites may result, in one or more deletions of the original cytoplasmic domain.
• cytoplasmic domain should retain, directly or indirectly, its inherent modifying enzyme activity activity, either by retaining a modifying enzyme activity binding site such as a Jak binding site, or by incorporating an active modifying enzyme activity in the cytoplasmic domain itself.
• Activation of the receptor and of the signaling pathway is achieved by binding of a ligand to the ligand-binding domain and by binding of a prey polypeptide to the heterologous bait polypeptide comprised in the cytoplasmic domain of the receptor.
• the gene, encoding the recombinant receptor comprising the bait polypeptide may be placed downstream either a constitutive or an inducible promoter.
• the latter construction may have some advantages in cases where there is a competition for the binding site between prey polypeptides and endogenous polypeptides.
• Induction of the recombinant receptor comprising the bait polypeptide in presence of the prey polypeptides may facilitate the binding and avoid saturation of the binding sites with endogenous polypeptides
• One preferred embodiment is a recombinant receptor according to the invention whereby the activation site is a phosphorylation site and the modifying enzyme activity is a kinase.
• Another preferred embodiment of the invention is a homomultimerizing recombinant leptin receptor, with a heterologous bait polypeptide fused into, or, preferentially, at the carboxyterminal end of its cytoplasmic domain.
• Said heterologous bait polypeptide may replace part of said cytopmasmic domain.
• the three conserved tyrosine phosphorylation sites of the cytoplasmic domain are inactivated, more preferentially by a replacement of tyrosine by phenylalanine.
• Another preferred embodiment is a homomultimerizing recombinant receptor in which an inactivated cytoplasmic domain of the leptin receptor, comprising a heterologous bait polypeptide, as described above, is fused to the ligand binding domain of the erythropoietin (EPO) receptor.
• EPO erythropoietin
• Still another embodiment is a heteromultimerizing recombinant receptor in which the inactivated cytoplasmic domain of the leptin receptor, comprising a heterologous bait polypeptide is fused to the lnterleukine-5 receptor ⁇ -chain ligand- binding domain for one subunit, and to the interleukine-5 receptor ⁇ -chain for another subunit.
• Still another embodiment is a heteromultimerizing recombinant receptor in which the inactivated cytoplasmic domain of the leptin receptor, comprising a heterologous bait polypeptide is fused to the GM-CSF ⁇ -chain ligand-binding domain for one subunit, and to the interleukine-5 receptor ⁇ -chain for another subunit.
• the bait is modified by the bait-mod ifying-enzyme activity which can be, but is not necessarily identical to the modifying enzyme activity which is modifying the activation site.
• the recombinant receptor can be a chimeric receptor, in which the ligand binding domain and the cytoplasmic domain are derived from two different receptors. Preferentially, the receptor is a multimerizing receptor.
• the cytoplasmic domain of the recombinant receptor comprises a heterologous bait polypeptide, which can be fused to the carboxyterminal end, or can replace a part of this carboxyterminal end or can be situated in the cytoplasmic domain itself, as an insertion or a replacement of an endogenous internal fragment.
• a heteromultimerizing receptor not all the chains need to comprise the bait, but it is sufficient if one of the composing chains does comprise the bait in its cytoplasmic domain.
• At least one of the activation sites in the cytoplasmic domain of the receptor has been inactivated, so that the receptor is not activated and there is no active signaling pathway if only a ligand is binding to the ligand-binding domain of said recombinant receptor.
• Such inactivation can be obtained in several ways, such as by replacement of the amino acid, which can be activated, by another amino acid, or by changing the amino acid context of the activation site or by deleting the activation site. Insertion of the heterologous bait polypeptide and inactivation of the activation sites may result in one or more deletions of the original cytoplasmic domain.
• cytoplasmic domain should retain, directly or indirectly, its inherent modifying enzyme activity, either by retaining a modifying enzyme binding site, or by incorporating an active modifying enzyme activity in the cytoplasmic domain itself.
• the activation site is a phosphorylation site
• the modifying enzyme activity is a kinase activity.
• the modification of the bait may be either in cis or in trans, i.e. by an enzymatic activity that is situated on the same cytoplasmic domain, or by an enzymatic activity that comes from elsewhere.
• the modification of the bait is induced by binding of a ligand to the ligand-binding domain.
• One preferred embodiment is a homodimerizing receptor in which the bait is phosphorylated by the inherent kinase activity of the cytoplasmic domain, preferentially a Jak kinase that is binding to said cytoplasmic domain.
• Another preferred embodiment is a heteromultimerizing receptor where the cytoplasmic domain of one chain comprises a bait to be modified, and the cytoplasmic domain of another chain comprises the bait-modifying enzyme activity.
• Activation of the receptor and of the signaling pathway is achieved by binding of a ligand to the ligand-binding domain and by binding of a prey polypeptide to the heterologous bait polypeptide situated in the cytoplasmic domain of the receptor. Binding of said prey polypeptide is dependent upon the modification state of said heterologous bait polypeptide, it means that binding occurs only in case the bait is modified or only in case the bait is not modified.
• prey polypeptide is a fusion protein comprising a polypeptide that can interact directly or indirectly with a bait polypeptide and another polypeptide that comprises at least one activation site.
• Said activation site is preferentially a phosphorylation site, more preferentially a tyrosine phosphorylation site.
• said tyrosine phosphorylation site is part of a Signal Transducer and Activator of Transcription (STAT) binding site, most preferentially part of a STAT1 and/or STAT3 binding site.
• STAT Signal Transducer and Activator of Transcription
• Direct interaction means that there is a direct protein-protein contact between the heterologous bait polypeptide and the prey polypeptide; indirect interaction means that the heterologous bait polypeptide interacts with one or more other polypeptides to form a complex that interacts with said prey polypeptide or vice versa.
• the prey polypeptide may interact either with only one or with several polypeptides from the complex.
• the binding of the prey polypeptide to the bait polypeptide may be dependent upon the modification state of said bait polypeptide and/or of proteins within the binding complex.
• the prey polypeptide may comprise a Nuclear Export Sequence (NES), to ensure that it is available in the cytosol.
• NES Nuclear Export Sequence
• the NES signal (amino acids 37-46) of the heat-stable inhibitor of the cAMP-dependent protein kinase has been shown to override a strong nuclear localisation signal (Wiley et al., 1999). This NES will keep the prey polypeptide in the cytoplasm even if it has a strong nuclear localisation signal, facilitating the interaction with the bait.
• One preferred embodiment is a prey polypeptide according to the invention, whereby said prey polypeptide interacts with the heterologous bait polypeptide of a recombinant receptor according to the invention.
• the activation site of the prey polypeptide can be modified by the modifying enzyme activity inherent to the cytoplasmic domain of the receptor. The modification of the activation site will activate the signaling pathway.
• said activation site is a phosphorylation site and the modifying enzyme activity is a kinase activity. More preferentially, this activation comprises binding of a STAT polypeptide to the phosphorylated phosphorylation site, followed by phosphorylation of said STAT polypeptide and subsequent dimerization of two phosphorylated STAT molecules.
• Another aspect of the invention is a vector, encoding a recombinant receptor according to the invention and/or a vector, encoding a prey polypeptide according to the invention.
• Said recombinant receptor and said prey polypeptide may be situated on one or on separated vectors.
• the vector can be any vector, know to the person skilled in the art, including but not limited to episomal vectors, integrative vectors and viral vectors.
• a preferred embodiment is a bait vector whereby the bait may be integrated in the chromosome by a recombinase-assisted integration such as cre-lox or flp-frt, and/or a retroviral prey vector that allows retroviral integration in the genome.
• Another aspect of the invention is an eukaryotic cell comprising a recombinant receptor according to the invention.
• the eukaryotic cell is obtained by transformation or transfection with one or more vectors according to the invention.
• Said eukaryotic cell comprises, but is not limited to yeast cells, fungal cells, plant cells, insect cells and mammalian cells.
• the eukaryotic cell is a mammalian cell.
• a preferred embodiment is an eukaryotic cell line expression the mouse retroviral receptor, allowing safe retroviral work using retroviral cDNA libraries.
• Still another aspect of the invention is a kit, comprising one or more cloning vectors allowing the construction of one or more vectors according to the invention.
• a cloning vector, encoding a recombinant receptor in which the part, encoding for the cytoplasmic domain comprises one or more restriction sites allowing an "in frame" fusion of a nucleic acid fragment encoding a polypeptide can easily be used to construct a vector encoding a recombinant receptor according to the invention.
• a cloning vector encoding a first polypeptide comprising at least one activation site, comprising one or more restriction sites allowing an "in frame" fusion of a nucleic acid encoding a second polypeptide with said first polypeptide can easily be used to construct a vector encoding a prey polypeptide according to the invention.
• other cloning strategies known to the person skilled in the art may be used.
• Still another aspect of the invention is a method to detect compound-compound binding using a recombinant receptor and/or a prey polypeptide according to the invention.
• an eukaryotic cell, carrying a recombinant receptor according to the invention is transformed or transfected with a vector library encoding prey polypeptides according to the invention. Bait-prey binding will result in an activation of the signaling pathway and can be detected by the use of a reporter system.
• the use of a chimeric receptor may represent an additional advantage for this method.
• a first advantage of the use of a chimeric receptor in this method is that it allows the elimination of a non bait-specific background.
• a difference can be made between bait-specific and non bait- specific binding.
• This can be realized by the use of a host cell carrying at least two receptors, a first receptor, comprising a first ligand binding domain and a cytoplasmic domain that does not comprise an activation site neither a heterologous bait polypeptide and a second receptor, comprising the same inactivated cytoplasmic domain, however with a heterologous bait polypeptide now, and a second ligand binding domain.
• a positive signal can only be detected when there is a non bait-specific interaction of a prey polypeptide fused to a polypeptide comprising an activation site with the cytoplasmic domain of the receptor; these cells can be selected and/or eliminated.
• the second ligand can be added to the medium.
• a positive signal will only be detected upon specific bait-prey interaction, as the preys binding to the cytoplasmic domain have been removed.
• One specific embodiment of the method to detect compound-compound binding is a method whereby said binding is a protein-protein interaction.
• Another specific embodiment is a method to detect protein-protein interaction, whereby said interaction is modification state dependent.
• Still another specific embodiment is a method to detect compound-compound binding, whereby said binding is mediated by three or more partners. In this case, one or more partners may not be or not completely be of proteineous nature.
• a recombinant receptor, according to the invention may, as a non-limiting example, bind to a small molecule.
• the prey polypeptide, according to the invention may also bind to the small molecule, so that bait and prey are linked together by said small molecule.
• Said small molecule may be present in the host cell, as a compound produced by the cell itself, or as a compound that is taken up from the medium.
• said method to detect compound-compound binding comprises the construction of an eukaryotic cell comprising a recombinant receptor according to the invention, followed by transformation or transfection of said cell by a library of prey polypeptide vectors according to the invention.
• the compound-compound binding is detected by the activation of the receptor, leading to an active signaling pathway, resulting in the induction of a reporter system.
• a reporter system can be any system that allows the detection and/or the selection of the cells carrying a recombinant receptor according to the invention. It is clear for the person skilled in the art that several reporter systems can be used.
• a luciferase gene, an antibiotic resistance gene or a cell surface marker gene can be placed after a promoter that is induced by the signaling pathway.
• reporter systems may be used that are based on the change in characteristics of compounds of the signaling pathway, when said pathway is active, such as the phosphorylation and/or dimerisation of such compounds.
• Receptor does not necessarily indicate a single polypeptide, but may indicate a receptor complex, consisting of two or more polypeptides, and comprising a ligand binding domain and a cytoplasmic domain.
• Recombinant receptor means that at least one of said polypeptides is recombinant.
• the polypeptide comprising the cytoplasmic domain is recombinant.
• Activation site of a receptor is the site that, in the wild type receptor, is modified after binding of a ligand to the ligand binding domain, leading to a reorganization of the receptor and subsequent activation of the modifying enzyme activity, and to which a compound of the signaling pathway can bind after modification, or any site that can fulfill a similar function.
• the activation site is not necessarily located on the same polypeptide as in the wild type receptor, but may be situated on another polypeptide of the receptor complex.
• Modifying enzyme activity as used here means the enzymatic activity, associated to or incorporated in the cytoplasmic domain of the receptor that is normally induced upon binding of the ligand to the ligand binding domain and subsequent reorganization of the receptor (e.g. by a conformational change), and may modify the activation site.
• the activation site is a phosphorylation site and the modifying enzyme activity is a kinase activity.
• the bait-modifying enzyme activity means the activity which modifies the bait. It can be, but is not necessarily identical to the modifying enzyme activity.
• Activation of a receptor means that the receptor is inducing a signaling pathway, by binding of a compound of the signaling pathway to the modified activation site, whereby said activation normally results in the induction or repression of one or more genes.
• Said gene is preferentially a reporter gene, which allows monitoring the activation of the receptor.
• An activated receptor is a receptor where the binding of a compound to the activation site has been enabled by modification of said site. A receptor in which the modifying enzyme activity has been induced, without modification of an activation site is not considered as activated.
• Multimerizing receptor as used here means that the activated receptor comprises several polypeptides. It does not necessarily imply that the multimerization is induced by ligand binding: the receptor can exist as a preformed complex of which the conformation is changed upon ligand binding.
• Polypeptide as used here means any proteineous structure, independent of the length and includes molecules such as peptides, phosphorylated proteins and glycosylated proteins. Polypeptide as used herein is not necessarily indicating an independent compound but can also be used to indicate a part of a bigger compound, such as a domain of a protein.
• Heterologous bait polypeptide, as comprised in the cytoplasmic domain of a receptor means that within the cytoplasmic domain, or fused to the cytoplasmic domain, there is a polypeptide that is not present in the cytoplasmic domain of the non-recombinant receptor. Said heterologous bait polypeptide may replace a part of said cytoplasmic domain. Bait herein means that this polypeptide can interact with other polypeptides, not belonging to the normal receptor complex.
• Prey polypeptide as used here means a fusion protein comprising a polypeptide that can bind with the heterologous bait polypeptide and a polypeptide that comprises at least one activation site.
• Ligand means every compound that can bind to the extracellular domain of a receptor and that is able to initiate the signaling pathway by binding to said extracellular domain. Initiating as used here means starting the events that normally directly follow the binding of the ligand to the extracellular domain of a receptor, e.g. multimerization for a multimerizing receptor, but it does not imply activation of the receptor and/or accomplishing of the signaling pathway.
• Compound means any chemical or biological compound, including simple or complex organic or inorganic molecules, peptides, peptido-mimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof.
• Bind(ing) means any interaction, be it direct or indirect.
• a direct interaction implies a contact between the binding partners.
• An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two compounds. This interaction can be completely indirect, with the help of one or more bridging compounds, or partly indirect, where there is still a direct contact that is stabilized by the interaction of one or more compounds.
• Functional fragment of the inactivated leptin receptor cytoplasmic domain means a fragment of the leptin receptor cytoplasmic domain that still allows binding of the Jak kinases.
• Inactivation of an activation site means any change, mutation or deletion that is inhibiting a modification at the position of the potentially modified residue in the polypeptide.
• inactivation of a tyrosine phosphorylation site means any change, mutation or deletion that is inhibiting a phosphorylation at the position of the potentially phosphorylated tyrosine residue in the polypeptide. Preferentially, it is a mutation at this position; more preferentially, it is a change of tyrosine into phenylalanine.
• Cloning vector is a vector that is generally considered as an intermediate step for the construction of another vector. It is intended to insert one or more nucleic acid fragments, in order to obtain one or more new vectors that will be used to transform or transfect the host cell of interest, or as cloning vectors themselves.
• Figure 1 Principle of the receptor-based interaction trap. Ligand binding leads to activation of a modifying enzyme activity (MA). Due to the inactivation of the normal receptor activation site (inactivated activation site, iAS), the activation of modifying enzyme activity does not result in an activation of the signaling pathway, unless the heterologous bait in the cytoplasmic domain of the recombinant receptor (indicated as 'bait') is binding to a prey polypeptide (indicated as 'prey') which is fused to a polypeptide comprising an activation site (AS). The modifying enzyme activity can now modify this activation site; modification (x) of this activation site results in activation of the signaling pathway and induction of a reporter system (indicated as 'detectable activity').
• MA modifying enzyme activity Due to the inactivation of the normal receptor activation site (inactivated activation site, iAS), the activation of modifying enzyme activity does not result in an activation of the signaling pathway, unless
• Figure 2 Functionality of EpoR-LepR chimera in the Hek293T PAP21 cell line, as measured by luciferase light emission, measured in a chemiluminescence counter (counts per second, cps).
• the cells were transfected with: a. pSV-SPORT + pMET7mcs + pGL3-rPAP1-luci + pUT651 b. pSV-SPORT EpoR/LepR + pMET7mcs + pGL3-rPAP1-luci + pUT651 c. pMET7 LepRY985/1077F + pMET7mcs + pGL3-rPAP1-luci +pUT651 NC: non-stimulated negative control. Stimulations were carried out as described in the examples.
• Figure 3 Functionality of p53-SV40 LargeT interaction trap, as measured by luciferase light emission, measured in a chemiluminescence counter (cps).
• the cells were transfected with: a. pSV-SPORT + P MG1-SVT + pGL3-rPAP1-luci + pUT651 b. pSV-SPORT + pMG1-CIS + pGL3-rPAP1-luci + pUT651 c. pSV-SPORT + pMET7-SVT + pGL3-rPAP1-luci + pUT651 d. pSEL1-p53 + pMG1-SVT + pGL3-rPAP1-luci + pUT651 e. pSEL1-p53 + pMG1-CIS + pGL3-rPAP1-luci + pUT651 f. pSEL1 -p53 + pMET7-SVT + pGL3-rPAP1 -luci + pUT651
• NC non-stimulated negative control. Stimulations were carried out as described in the examples.
• Figure 4 Functionality of the EpoR-CIS phosphorylation-dependent interaction trap, as measured by luciferase light emission, measured in a chemiluminescence counter (cps).
• the cells were transfected with: a. pSV-SPORT + pMG1-CIS + pGL3-rPAP1-luci + pUT651 b. pSV-SPORT + pMG1-SVT + pGL3-rPAP1-luci + pUT651 c. pSV-SPORT + pEF-FLAG-l/mCIS + pGL3-rPAP1-luci + pUT651 d. pSEL1-EpoR + pMG1-CIS + pGL3-rPAP1-luci + pUT651 e.
• FIG. 5 Functionality of the IRS1-GRB2-Vav indirect interaction trap, as measured by luciferase light emission, measured in a chemiluminescence counter (cps).
• the cells were transfected with: a. pMET7mcs + pMG1-CIS + pGL3-rPAP1-luci + pUT651 b. pMET7mcs + pMG1-GRB2S + pGL3-rPAP1-luci + pUT651 c. pMET7mcs + pMG1-VavS + pGL3-rPAP1-luci + pUT651 d.
• FIG. 6 Functionality of the IRS1-GRB2-Vav indirect interaction trap, as measured by luciferase light emission, measured in a chemiluminescence counter (cps): GRB2 dose dependent inhibition of the signal.
• the cells were transfected with: a. pMET7 LepR-IRS1 + 200 ng pMG1-VavS + pGL3-rPAP1-luci + pUT651 b. pMET7 LepR-IRS1 + 200 ng pMG1-VavS + 200 ng pMET7 GRB2SH3 + pGL3- rPAPI-luci + pUT651 c. pMET7 LepR-IRS1 + 200 ng pMG1-VavS + 1000 ng pMET7 GRB2SH3 + pGL3- rPAP1-luci + pUT651
• NC non-stimulated negative control. Stimulations were carried out as described in the examples.
• Figure 7 Functionality of the IRS1-GRB2-Vav indirect interaction trap, as measured by luciferase light emission, measured in a chemiluminescence counter (cps): VavS dose dependent inhibition of the signal.
• the cells were transfected with: a. pMET7 LepR-IRS1 + 200 ng pMG1-VavS + pGL3-rPAP1-luci + pUT651 b. pMET7 LepR-IRS1 + 200 ng pMG1-VavS + 200 ng pMET7 VavS + pGL3-rPAP1- luci + pUT651 c. pMET7 LepR-IRS1 + 200 ng pMG1-VavS + 1000 ng pMET7 VavS + pGL3-rPAP1- luci + pUT651 NC: non-stimulated negative control. Stimulations were carried out as described in the examples.
• Figure 8 Layout of an optimised MAPPIT-based two-hybrid screening method The procedure encompasses three successive steps, indicated with encircled numbers. First, cells expressing the chimeric receptor with the C-terminal "bait" (CR- Bait) are generated upon recombinase-assisted genomic integration, followed by hygromycin selection. Next, gp130-"prey” chimeras are expressed upon retroviral gene transfer. Finally, if cognate "bait"-"prey” interaction occurs, ligand binding induces a signalling cascade leading to induction of the puromycin resistance marker and concomitant formation of cell colonies in selective medium. Direct RT-PCR amplification of "prey" encoding transcripts from lysed cell colonies allows rapid "prey” identification.
• Figure 9 the MAPPIT procedure for two-hybrid screening
• the HEK293-16 cell line shows ligand-induced puromycin resistance.
• HEK293-16 cells were seeded in a 24 well plate, and were left untreated (top well), or were subject to puromycin selection (1 ⁇ g/ml) with (middle well) or without (bottom well) prior activation of gp130 using LIF for 48 hours. After 1 week, surviving cells were stained with crystal violet.
• HEK293-16 cells expressing the EpoR"bait.
• HEK293-16 cells were co-transfected with the pcDNA5/FRT-EpoR"bait” and the Flp recombinase expression vectors and selected for hygromycin resistance (100 ⁇ g/ml) for 10 days. Cells were stained using polyclonal antiserum recognising the extracellular domain of the EpoR and Alexa488-labelled secondary antibody. Solid and dotted lines show parental or hygromycin-selected HEK293-16 cells, respectively.
• C Selection of cells based on a cognate "prey"-"bait" interaction.
• Hygromycin- resistant cells from (B) were seeded in a 24 well plate, infected with CIS"prey” expressing retrovirus (1/30 dilution of retroviral stock) for 48 hours, and were either left untreated (top well) or were stimulated with Epo for another 48hours (middle well), prior to treatment with puromycin (1 ⁇ g/ml).
• the bottom well shows Epo-stimulated cells selected as above, but expressing irrelevant lacZ protein (1/3 dilution of retroviral stock). Surviving cells were stained after 7 days with crystal violet.
• Puromycin-resistant cells express the "prey" chimera.
• Parental HEK293-16 cells or puromycin-resistant cells from (C) were permeabilized, sequentially treated with anti- FLAG antibody and FITC-labelled secondary antibody and subjected to FACS analysis. Solid and dotted lines show parental or puromycin-selected HEK293-16 cells, respectively.
• (INSET) RT-PCR detection of transcripts encoding the "prey" chimera. Cells from (C) were lysed and the "prey"-encoding transcript was amplified by RT- PCR. The arrow indicates the CIS-specific amplicon, which was verified by DNA sequencing. A negative control on parental cells was also performed (middle lane). M: marker lane.
• HEK293T cells were transiently transfected with expression constructs for the EpoR"bait" (pSEL1-EpoR) and for the SOCS-2"prey".
• the middle panel and lower panel respectively show ligand-dependent "prey” and STAT3 phosphorylation which is only observed upon transfection with pSEL1-EpoR but not with pSELI-EpoRF.
• Expression controls for the SOCS-2"prey" and for STAT3 are also shown.
• Figure 10 Functionality of IL3R-, IL5R- and GM-CSFR-LepR chimera in the Hek 293T cell line, as as measured by luciferase light emission, measured in a chemiluminescence counter (cps).
• the cells were transfected with: a. pSV-SPORT-EpoR/LepR + pGL3-rPAP1-luci + pUT651 b. pSV-SPORT-IL-3R ⁇ /LepR + pSV-SPORT- ⁇ rJLepR + pGL3-rPAP1-luci + pUT651 c.
• NC non-stimulated negative control. Stimulation was as described in the examples.
• Figure 11 Functionality of the Smad3-Smad4 phosphorylation-dependent interaction trap, as measured by luciferase light emission, measured in a chemiluminescence counter (cps).
• the cells were transfected with: a. pSV-SPORT- ⁇ c /LepR-F3-ALK4CA + pSV-SPORT-GM-CSFR ⁇ /LepR-F3-Smad3 + pMG2-Smad4 + pXP2d2-rPAP1luci + pUT651. b. pSV-SPORT- ⁇ c /LepR-F3-Smad3 + pSV-SPORT-GM-CSFR ⁇ /LepR-F3-ALK4CA + pMG2-Smad4 + pXP2d2-rPAP1luci + pUT651. c.
• Recombinant mouse leptin, recombinant human leukemia inhibitory factor (LIF) and recombinant human erythropoietin (Epo) were all purchased from R&D Systems. Typical stimulation conditions were 100ng/ml leptin, 1 ng/ml LIF and 50 ng/ml Epo.
• ⁇ NX-Eco cells were seeded at a density of 6x10 6 cells/petridish the day prior to transfection. Cells were transfected with 50 ⁇ g of the retroviral vector pBG1- CIS according to the calcium phosphate procedure. 25 ⁇ M chloroquine was added 5 min.
• target cells were seeded at a density of 2x10 4 cells/well in a 24-well plate, and 10 6 in 75cm 2 culture flasks. The day after, cells were incubated for 24-48 hours with supernatant containing virus, diluted in medium as indicated. Polybrene (Sigma) was added at a final concentration of 2.5 ⁇ g/ml. After infection, cells were stimulated with Epo (50 ng/ml) for 24-48 hours, followed by puromycin (1-2 ⁇ g/ml as indicated; Sigma) selection for 10 days.
• Epo 50 ng/ml
• puromycin 1-2 ⁇ g/ml as indicated; Sigma
• PCR polymerase chain reactions
• Pfu polymerase Pfu polymerase
• the mouse leptin receptor (LepR) transmembrane and intracellular parts were amplified by PCR using forward primer MBU-O-447 that contains a Pad restriction enzyme recognition site and the reverse primer MBU-O-448 that contains both a linker sequence (Gly-Gly-Ser) and a multi cloning site (MCS) with Sail, Sad, Spel, Notl and Xbal recognition sites. Only one amino acid from the extracellular part of the LepR was included in the fragment (Gly).
• Primer design resulted in the insertion of an Asn between the Pad generated Leu-lle sequence and the extracellular Gly.
• the amplicon was gel-purified and ligated in the pCR ® -Blunt vector (Invitrogen). Pacl-Sacl digestion on this pCR ® -Blunt construct results, after gel-purification, in the desired LepR fragment.
• RT-PCR was performed as follows: 2 ⁇ l (2 ⁇ g) of oligodT (12-18 mer; Pharmacia) was added and incubated at 70°C for 10 min., the reaction mixture was chilled on ice for 1 min., cDNA was prepared by adding 4 ⁇ l of 10x RT buffer (Life Sciences), 1 ⁇ l 20 mM dNTP's (Pharmacia), 2 ⁇ l 0,1M DTT, and 1 ⁇ l of MMLV reverse transcriptase (200U; Superscript RT; Life Technologies) to an end volume of 20 ⁇ l. Incubations were as follows: RT for 10 min., 42°C for 50 min., 90°C for 5 min., and 0°C for 10 min.
• RnaseH 2U ; Life Technologies
• RnaseH 2U ; Life Technologies
• PCR on this cDNA was performed using Pfu enzyme (5 U; Stratagene).
• Forward primer (MBU-O-167) and reverse primer (MBU-O- 308) were designed to amplify the extracellular part of the EpoR (amino acids 1-249) between a Kpnl and Pad site.
• a band of correct size was purified and the DNA was digested with Kpnl and Pad and was inserted into the Kpnl-Pacl opened pSV-SPORT- IL-5R ⁇ /IFNaR2-2 vector.
• This vector contains a chimeric receptor that has the extracellular domain of the IL-5R ⁇ receptor, fused to the transmembrane and intracellular domains of lFNaR2-2.
• a Pad site was added to the fusion point by means of the QuikchangeTM site-directed mutagenesis kit (Stratagene, La Jolla) which resulted in the insertion of two amino acids (Leu-lle) before the most membrane-proximal, extracellular amino acid (Lys) of IFNaR2-2.
• the extracellular domain of IL-5R ⁇ could be exchanged by the one of EpoR, as described above.
• the LepR fragment generated by Pad-Sad digestion was ligated in the Pad-Sad digested and gel-purified pSV-SPORT-EpoR/IFNaR2-2 vector, resulting in pSV- SPORT-EpoR/LepR.
• the pSV-SPORT-IL-5R ⁇ /IFNaR2-2 and pSV-SPORT- ⁇ c /IFNaR1 vectors express an IL-5R ⁇ /IFNaR2-2 and a ⁇ c /IFNaR1 chimera, respectively, composed of the extracellular portion of the IL-5R ⁇ or ⁇ c chain, and the transmembrane and intracellular parts of the IFNaR2-2 or IFNaRI .
• a Pad site was used to generate the fusion site just preceding the transmembrane segment.
• the IFNaR2-2 or IFNaRI parts in these vectors were replaced by the same segments of the LepR, using the Pad site and an Xbal site which is located just after the IFNaR2-2 or IFNaRI stop codon. Therefore, the LepR fragment was generated by a Pad-Xbal digest of the pSV-SPORT- EpoR/LepR vector (see example 1), and was inserted into the Pad-Xbal opened and gel-purified pSV-SPORT-IL-5R /IFNaR2-2 and pSV-SPORT- ⁇ c/IFNaR1 vectors, resulting in the vectors pSV-SPORT-IL-5R ⁇ /LepR and pSV-SPORT- ⁇ c/LepR.
• the pSV-SPORT-IL-3R ⁇ /LepR and pSV-SPORT-GM-CSFR ⁇ /LepR vectors were constructed as follows: the extracellular portion of the IL-3R ⁇ and GM-CSFR ⁇ chains were amplified using standard RT-PCR procedures with Pfu polymerase. 2 ⁇ l TF-1 cDNA was used as input. Forward primers were MBU-O-752 (IL-3R ⁇ ) and MBU-O-754 (GM-CSFR ⁇ ), and generated a Kpnl site. Reverse primers MBU-O-753 (IL-3R ⁇ ) and MBU-O-755 (GM-CSFR ⁇ ), contain a Pad site allowing in frame fusion to the LepR.
• the Kpnl-Pacl excised extracellular fragments were ligated into the Kpnl-Pacl opened pSV-SPORT-IL-5R ⁇ /LepR.
• pSV-SPORT-IL-5R ⁇ /LepR For the GM-CSFR ⁇ construction, a partial Kpnl digest was applied since the extracellular portion contained an internal Kpnl site.
• the resulting vectors, pSV-SPORT-IL- 3R ⁇ /LepR and pSV-SPORT-GM-CSFR ⁇ /LepR contain chimeric receptors composed of the extracellular portion of the IL-3R ⁇ or GM-CSFR ⁇ chain fused to the transmembrane and cytoplasmatic tail of the LepR.
• mutant leptin receptors (Eyckerman et al., 1999) Y985-1077F and Y985-1077- 1138F (LepR-F3; previously called F-all) were generated using the QuikchangeTM site- directed mutagenesis procedure using Pfu polymerase (Stratagene) on the pMET7- LepR template.
• Mutagenic oligonucleotides were MBU-O-157, MBU-O-158, MBU-O- 159, MBU-O-160, MBU-O-161 and MBU-O-162. Each single mutation was coupled to a change in restriction cleavage and was confirmed by restriction and DNA sequence analysis. The double and triple mutants were created using a sequential approach.
• pMET7mcsA primers MBU-O-567 and MBU-O-568.
• the pMET7mcs vector is a modified version of pMET7 containing an expanded MCS by insertion of the extra unique Bglll, EcoRV, BstEII, Agel and Xhol restriction sites.
• the forward primer contains from 5' to 3' an Apal restriction site, a Kozak consensus sequence, a flag-tag encoding sequence (Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-lle), and a Bglll restriction site.
• the reverse primer encodes an additional hinge sequence (Gly-Gly-Ser) and contains an EcoRI recognition site.
• Apal and EcoRI digestion of the PCR product (after subcloning in pCR ® -Blunt) and of pMET7-mcsA, allowed us to ligate the gp130 fragment into the pMET7 vector, generating the pMET7-flag-gp130 construct.
• SV40 largeT antigen (SVT) was amplified using a vector from the HybriZAP-2.1 Two- Hybrid cDNA synthesis kit (Stratagene, pSV40) as template.
• Primers MBU-O-445 and MBU-O-446 were used to generate a DNA fragment encoding 448 amino acid between residues 261 and 708.
• the N-terminal deletion eliminates the nuclear targeting signal in SVT.
• the forward primer contains an EcoRI recognition site that allows in-frame ligation to the gp130-hinge sequence.
• the reverse primer contains additional Nrul, Xhol, Bglll, Notl and Xbal restriction sites and also encodes the stop codon after the SVT coding sequence.
• a DNA fragment encompassing murine p53 was amplified with MBU-O-450 and MBU- O-451 using the p53 control plasmid from the HybriZAP-2.1 Two-Hybrid cDNA synthesis kit (Stratagene) as template.
• the forward primer contains a Sail restriction site that allows in-frame coupling to the EpoR/LepR-F3 hinge construct.
• the reverse primer contains a STOP codon and a Xbal restriction site.
• the 243 amino acid-long p53 fragment (amino acids 73-315) contains the interaction site with SVT, but lacks the nuclear targeting signal and the oligomerisation domain.
• RT-PCR was performed using standard reaction conditions as described in section 1 .1.1.
• An intracellular fragment of the human EpoR (amino acids 370-453) was amplified from 4 ⁇ l TF1 cDNA using MBU-O-675 and MBU-O-676 as forward and reverse primer respectively, with two consecutive PCR reactions and an intermediate gel-purification. Sad and Xbal recognition sites are present in the forward and reverse primers respectively.
• the reverse primer also encodes a stop codon.
• the fragment was subcloned in pCR ® -BIunt, digested with Sstl (which has the same recognition site as Sad) and Xbal, and ligated into Sstl-Xbal digested and gel-purified pSEL1 vector, resulting in the pSEL1-EpoR construct.
• the complete coding region for mouse Cytokine Inducible SH2-containing protein CIS was amplified using MBU-O-677 and MBU-O-678 as forward and reverse primer respectively.
• the forward primer contains an EcoRI recognition site and the reverse primer contains a Xbal recognition site and the stop codon.
• the amplified and gel-purified fragment was subcloned into the pCR ® -Blunt vector.
• the insert was recovered by EcoRI and Xbal digestion and gel-purification and was cloned into an EcoRI-Xbal digested and gel-purified pMG1-SVT vector, leading to the pMG1- CIS vector.
• the SH2 domain of GRB2 (aa 60-158) was amplified in the same way using primers MBU-O-469 and MBU-O-470 as forward and reverse respectively.
• the forward primer contains an extra EcoRI recognition site which allows in frame fusion to the gp130 chain
• the reverse primer contains an extra stop codon and a Xbal enzyme recognition site.
• a fragment of human GRB2 comprising the C-terminal SH3 domain (aa 159-217) was amplified using the pMG1-GRB2 construct as template and MBU-O-770 and MBU-O- 468 as forward and reverse primer respectively.
• MBU-O-770 allows in frame fusion to the flag tag by a Bglll site
• MBU-O-468 is described above.
• the GRB2 fragment was inserted in the pMG1 vector by a Bglll-Xbal based exchange, resulting in the pMET7-GRB2SH3 vector.
• VavS aa 259-789
• VavS human TF1 cell line by standard RT-PCR techniques.
• Primers were MBU-O-737 and MBU-O-738 as forward and reverse respectively.
• MBU-O-737 contains an extra EcoRI allowing in frame fusion to gp130, and MBU-O-738 contains a Stop codon and a Xhol enzyme recognition site.
• the amplified fragment was subcloned in the pCR ® -Blunt vector and ligated in the pMG1 vector through an EcoRI- Xhol based exchange.
• the VavS fragment was also amplified using forward primer MBU-O-771 , reverse primer MBU-O-741 and pMG1-VavS as template.
• the forward primer contains a BamHI site, which allows in frame fusion to the flag tag, and the reverse primer contains a stop codon and a Xbal restriction site.
• the amplicon was subcloned in the pCR ® -Blunt vector, and excised with BamHI and Xbal.
• the purified fragment was cloned in a Bglll-Xbal cut pMG1 vector, resulting in the pMET7-VavS construct. Construction of the pGL3-rPAP1-luci and pSEAP-rPAP1 reporter constructs
• Genomic DNA was isolated from the rat pheochromocytoma PC12 cell line using the DNAzol procedure (Gibco BRL).
• Optimal primers for PCR amplification of the rat PAP1 promoter were selected using the "Oligo Primer 3" program (http://www- genome.wi.mit.cdu/cgi-bin/primer3.cgi).
• Forward and reverse primers were MBU-O- 222 and MBU-O-223 respectively.
• Amplification was performed using Taq polymerase in 30 cycles: 2' at 94°C, 2' at 57°C, 2' at 72°C, followed by a 10' filling-in reaction at 72°C.
• Optimal MgCI 2 concentration was determined to be 6mM.
• the promoter fragment was cloned after polishing with Klenow polymerase in the pCR ® -Blunt vector.
• the promoter fragment was cut from this plasmid construct using a successive Pstl digest, Klenow treatment to polish this end, and BamHI digest, resulting in a blunt- sticky fragment.
• the gel-purified fragment was cloned into a Smal-Bglll opened and gel-purified pGL3 control vector (Promega). Digestion with Sstl-Spel restriction enzymes and gel-purification resulted in a fragment that was cloned into a Sstl-Nhel and purified pGL3 basic vector, resulting in the pGL3- rPAP1-luci construct.
• the full-length rPAP1 promoter fragment was excised from pGL3 rPAP1-luci using partial digestion with Kpnl and Xhol and ligated into the Kpnl-Xhol opened pXP2d2 vector (gift from Prof. S. Nordeen), resulting in pXP2d2-rPAP1-luci.
• the coding sequence for puromycin was amplified using primers MBU-O-719 and MBU-O-720 on the plRESpuro2 (Clontech) template.
• Insertion of the EpoR-LR-F3-EpoR into the pcDNA5/FRT vector was obtained by re- amplifying the complete chimeric construct using MBU-O-167 and MBU-O-769 on the pSEL1-EpoR template, followed by subcloning using Kpnl and Notl sites. This construct was named pcDNA5/FRT-EpoR.
• the SVT fragment was re-amplified from pMG1-SVT using forward primer MBU-O-766 and MBU-O-446.
• BamHI-Notl digestion allowed insertion in the pBMN-Z retroviral vector (gift from G. Nolan), resulting in vector pBG1-SVT.
• EcoRI-Notl based exchange of the SVT fragment for CIS resulted in the pBG1-CIS vector.
• the E. coli control of cell death gene (ccdB) was amplified using primers MBU-O-835 and MBU-O-836 and template pENTRYH (Life Technologies), and cloned in the pBG1-CIS vector by an EcoRI-Notl restriction-based insertion, resulting in the pBG1-ccdB vector.
• the LepR-F3 fragment was generated by a Pacl-Notl digest of the pSEL1 construct, gel-purified and inserted into the Pacl-Notl opened and gel-purified pSV-SPORT-GM-CSFR ⁇ or pSV-SPORT- ⁇ c vectors, resulting in the vectors pSV-SPORT-GM-CSFR ⁇ /LepR-F3 or pSV-SPORT- ⁇ c /LepR-F3.
• blunt ended vectors were incubated with Alkaline Phosphatase (Boehringer Mannheim) to dephosphorylate the blunt ends.
• Alkaline Phosphatase Boehringer Mannheim
• a construct containing the mouse cytoplasmic tail of ALK4 with mutation T206D, resulting in a constitutive active kinase, in the vector pGBT9 was obtained from Prof. D. Huylebroeck.
• the mutated cytoplasmic tail of ALK4 was removed from the construct by an EcoRI-BamHI digestion, gel-purified and incubated with Klenow fragment to polish the ends.
• This insert was ligated in the opened pSV- SPORT-GM-CSFR ⁇ /LepR-F3 and pSV-SPORT- ⁇ c/LepR-F3 vectors resulting in pSV- SPORT-GM-CSFR ⁇ /LepR-F3-ALK4CA and pSV-SPORT- ⁇ c/LepR-F3-ALK4CA.
• a construct containing a human cDNA encoding the entire Smad3 protein in vector pcdef was a kind gift of Prof. D. Huylebroeck.
• the Smad3 insert was removed with an EcoRI-Xhol digestion, gel-purified and the ends were polished by Klenow fragment (Boehringer Mannheim).
• This Smad3 insert was ligated into the opened pSV- SPORT-GM-CSFR ⁇ /LepR-F3 and pSV-SPORT- ⁇ c/LepR-F3 vectors (described above) resulting in pSV-SPORT-GM-CSFR ⁇ /LepR-F3-Smad3 and pSV-SPORT- ⁇ ⁇ JLepR-F3- Smad3.
• Generation of the pMG2-prey chimera For construction of the pMG2 vector, the pMG1-SVT vector was digested with EcoRI and Notl, followed by incubation with Klenow fragment (Boehringer Mannheim) to polish the ends.
• This blunt ended vector was incubated with Alkaline Phosphatase (Boehringer Mannheim) to dephosphorylate the blunt ends.
• Cassette rfB of the Gateway Vector Conversion System (Life Technologies) was then ligated into the opened vector leading to the pM ⁇ l -gateway vector.
• a PCR reaction using primers MBU-O-1094 and MBU-O-1076 on the pMG1-SVT template was performed, resulting in a fragment that contains gateway recombination sites. This fragment also contains a part (amino acids 905 - 918) of the gp130 chain.
• the fragment was then cloned in the pMG1 -gateway vector using a two-step gateway reaction (Lifetechnologies), resulting in pMG2-SVT, a prey construct with a total of 6 STAT recruitment sites.
• the pMG2- SVT construct was digested by EcoRI-Xhol and the vector was gel-purified.
• the prey we obtained a construct from Prof. D. Huylebroeck containing a cDNA encoding almost the entire human Smad4 protein, only lacking the first 3 amino acids.
• the Smad4 insert was removed by an EcoRI-Xhol digestion, gel-purified and ligated into the opened pMG2 vector.
• the Blasticidin system (Invitrogen) was used to create a stable cell line with an endogenous pSEAP-rPAPI reporter construct. Sensitivity to the toxic agent Blasticidin (Invitrogen) of Hek293T cells was estimated to be 3 ⁇ g/ml. 10 6 cells were seeded in a petridish and transfected the day after seeding using the Calcium Phosphate Transfection System (Life Technologies) according to the manufacturers instructions. A total of 20 ⁇ g DNA was transfected (18 ⁇ g of pSEAP-rPAP1seap and 2 ⁇ g of pcDNA6/V5-HisA, which contains a Blasticidin resistance gene).
• the transfected cells were seeded in 96 well plates at 10 cells per well. After 24 hours, Blasticidin was added at a concentration of 3 ⁇ g/ml and cells were maintained under selective conditions for 3-4 weeks. The resulting single cell clones were screened by stimulation for 24 hours with 20 ng/ml hyper-IL6 (fusion protein of IL-6 with its specific receptor IL6-R ⁇ ; Fischer et al., 1997). Hek293T PAP21 was selected as the best responsive cell line.
• hyper-IL6 fusion protein of IL-6 with its specific receptor IL6-R ⁇ ; Fischer et al., 1997.
• HEK293-16 cell line Flp-ln-293 cells were stably transfected with a plasmid containing expression cassettes for the mouse ecotropic retroviral receptor (mEcoR) and for neomycin resistance.
• mEcoR mouse ecotropic retroviral receptor
• neomycin resistant cells resistant to 400 ⁇ g/ml geneticin, Life Technologies
• the pool of neomycin resistant cells were supertransfected, at a ratio of 5:1 respectively, with the following two plasmids: i) a plasmid carrying the cDNA encoding the puromycin resistance marker (puromycin-N-acetyl-transferase) under control of promoter sequences of rPAP1 (pXP2d2-rPAP1-puro R ), ii) a plasmid carrying the cDNA for the blasticidin resistance marker (blasticidin S deaminase), under the control of the EM7 promoter (pcDNA6/V5-His, Invitrogen).
• cells were lysed in 100 ⁇ l RLT buffer (RNeasy ® method, Qiagen) and chromosomal DNA was sheared using Qiashredder columns (Qiagen).
• Beads were pre-treated according to the manufacturers' instructions (Dynabeads M- 280 Streptavidin, Dynal). Briefly, Dynabeads were washed twice in a high salt buffer (1M NaCI, 10mM Tris HCI pH7,5 and 1mM EDTA), and were incubated with 200 pmoles of biotinylated oligonucleotide directed against the gp130 chain (5' GGGCTGGGTAGACTCGGATCTTGAGAAGAC).
• beads were washed three times in the above mentioned high salt buffer and resuspended in a low salt buffer (0,15M NaCI, 10mM Tris HCI pH7,5 and 1mM EDTA) to a concentration of 10 ⁇ g/ ⁇ l. 5 ⁇ l of this suspension was added to 100 ⁇ l total lysate diluted 1/5 in the high salt buffer. 15' minutes after gentle rotation at room temperature, beads were washed three times with low salt buffer and eluted in 30 ⁇ l water for 2' at 65°C. 15 ⁇ l of this sample was used as input for a standard RT-PCR reaction with the Qiagen OneStep RT-PCR Kit.
• Primers 5' GGCATGGAGGCTGCGACTG and 5' TCGTCGACCACT GTGCTGGC were used for amplification of the "prey" fragment.
• efficient amplification was obtained with lysate from less than 10 3 cells.
• Luciferase was measured after lysis of the cells and addition of luciferase substrate (Luciferin, Duchefa). Light emission was measured using a TopCount chemiluminescence counter (Canberra Packard). All luciferase measurements were normalized using an expression construct constitutively expressing ⁇ -galactosidase (pUT651), which was measured in triplicate for every transfection using the GalactoStar kit (TROPIX).
• HEK293 library was generated using standard procedures. Briefly, 5 ⁇ g of HEK293 polyA+mRNA was used as input for both oligo-dT and random primed first strand synthesis with Superscript II reverse transcriptase (Life Technologies). Both the oligo-dT and random primers contain a Notl site. After second strand synthesis, adaptors containing an EcoRI site were ligated. The cDNA was analyzed using agarose gel electrophoresis and fragments between 0.5 and 2.5 Kbp were cloned unidirectionally in the pBG1-ccdB vector opened with EcoRI-Notl.
• a total of 6.10 7 "bait" expressing cells were seeded at a density of 2.10 6 per 175 cm 2 tissue culture flasks. 24 hours after seeding, cells were infected with the retroviral HEK293"prey" cDNA library (complexity of 2.10 6 ) for another 24 hours. After infection, cells were stimulated for 6,5 hours with 50 ng/ml Epo, and puromycin was added at a final concentration of 2 ⁇ g/ml for 20 days. Single cell colonies were picked and analysed using a functional assay and RT-PCR sequencing.
• Example 1 Functionality of EpoR-LepR chimera in the Hek293T PAP21 cell line
• 3 combinations of plasmids were transfected into Hek293T PAP21 cells: a. pSV-SPORT + pMET7mcs + pGL3-rPAP1-luci + pUT651 b. pSV-SPORT EpoR/LepR + pMET7mcs + pGL3-rPAP1-luci + pUT651 c.
• Forskolin is a chemical agent that activates the adenylate cyclase that is present within the cells, which leads to heightened levels of the second messenger cAMP.
• Treatment of transfected cells with forskolin alone did not result in a significant induction of luciferase activity.
• cAMP elevation leads to strong co-stimulation with the leptin signal on PAP1 induction (Eyckerman et al., 1999).
• the cells were lysed in the wells and luciferase substrate (Luciferin, Duchefa) was added. Light emission was measured using a TopCount chemiluminescence counter (Canberra Packard).
• the mock control transfection showed no signal in all cases. Results are shown in figure 2.
• the transfection with the EpoR/LepR chimera resulted in a 3,7 fold induction with erythropoietin, and a 6,5 fold induction with erythropoietin and forskolin. No significant signal was detected when stimulated with leptin, nor with leptin + forskolin.
• the LepR Y985/1077F mutant a 33,2 fold induction was detected when stimulated with leptin, and a 37,6 fold induction was detected when co-stimulated with forskolin. No signal was detected in both erythropoietin and erythropoietin + forskolin stimulated cells. All results were normalized using the internal transfection control vector pUT651 and the GalactoStar kit (See above).
• EpoR/LepR and LepR Y985/1077F The difference in induction between EpoR/LepR and LepR Y985/1077F is very likely due to the elimination in the latter receptor construct of tyrosines involved in the recruitment of tyrosine phosphatases and SOCS proteins to the complex, leading to an enhanced signal (Eyckerman et al., 1999).
• Example 2 Functionality of p53-SV40 LargeT interaction trap
• pSV-SPORT + pMG1-SVT + pGL3-rPAP1-luci + pUT651 b.
• a 300 ⁇ l precipitation mixture was prepared which contained 3.1 ⁇ g DNA (0.1 ⁇ g of pUT651 , 1 ⁇ g of each of the others). 200 ⁇ l was added to the cells for 6 hours after which they were washed once with Dulbecco's PBS. After washing, DMEM medium was added to the cells. After 24 hours cells were resuspended using 200 ⁇ l Cell Dissociation Agent which was neutralized with 2200 ⁇ l DMEM medium. Of this cell suspension 40 ⁇ l was brought into a 96 well plate for each transfection and stimulation was performed in triplicate.
• DMEM 60 ⁇ l DMEM was added to an end volume of 100 ⁇ l and 24 hours later, cells were stimulated for 24 hours with erythropoietin or erythropoietin plus forskolin (same concentrations as described above). A non-stimulated negative control was also included in the experiment. Luciferase measurements are shown in figure 3.
• Transfected cells from transfections a, b and c showed no significant induction of the reporter construct under all conditions tested.
• a 9,4 fold induction and a 14,6 fold induction was detected in transfected cells from transfection d, after stimulation with erythropoietin and erythropoietin plus forskolin respectively, implying an interaction- dependent signal.
• No signal was detected in transfection e and f. This implies a specific interaction, which leads to gp130-dependent STAT-3 activation. All results were normalized using the internal transfection control vector pUT651 and the GalactoStar kit (See above).
• EpoR-CIS phosphorylation-dependent interaction trap To determine the functionality of the EpoR-CIS phosphorylation-dependent interaction trap, the following plasmid combinations were transfected in 4x10 5 Hek293T cells, which were seeded the day before transfection: a. pSV-SPORT + pMG1-CIS + pGL3-rPAP1-luci + pUT651 b. pSV-SPORT + pMGI-SVT + pGL3-rPAP1-luci + pUT651 c. pSV-SPORT + pEF-FLAG-l/mCIS + pGL3-rPAP1-luci + pUT651 d.
• a precipitation mixture of 300 ⁇ l was prepared which contained 3,1 ⁇ g DNA (0,1 ⁇ g of pUT651 , 1 ⁇ g of each of the others). 200 ⁇ l of this mixture was applied to the cells. After 6 hours, the cells were washed once with Dulbecco's PBS, and DMEM medium was added. After 48 hours the cells were resuspended using 250 ⁇ l Cell Dissociation Agent. After neutralization with 2200 ⁇ l DMEM medium, 100 ⁇ l of this cell suspension was brought into a 96 well plate (Costar). The cells were stimulated with erythropoietin or erythropoietin plus forskolin (for final concentrations, see above).
• Luciferase expression was measured 24 hours after stimulation using a TopCount chemiluminescense counter (Canberra Packard).
• Transfected cells from transfections a, b, c, e, f and g showed no significant induction of luciferase activity.
• Transfected cells from transfection d showed a 6,2 fold and a 10,5 fold induction with erythropoietin or erythropoietin plus forskolin, respectively. This indicates an erythropoietin-dependent phosphorylation of the EpoR bait, resulting in interaction between the CIS protein and EpoR. Interaction leads to gp130 phosphorylation, STAT activation and thus signalling toward the rPAP1 promoter, leading to luciferase activity ( Figure 4).
• the 300 ⁇ l precipitation mixture contained also 0,05 ⁇ g of pUT651 DNA and 1 ⁇ g of pMET7 LepR-IRS1 and pGL3-rPAP1-luci DNA. From the normalized results ( Figure 7) we can conclude that gp130 in the gp130-VavS fusion construct is essential for PAP1 promoter induction since dose dependent competition with uncoupled VavS leads to a significant reduction in luciferase activity.
• Example 5 optimalization of the method for library screening
• a HEK293 cell clone was used (i) containing a FRT integration cassette in a transcriptionally active locus (Flp-ln-293 cell line, Invitrogen), (ii) stably expressing the murine ecotropic retroviral receptor EcoR, and (iii) with a stably integrated pXP2d2-rPAP1-puro R selection cassette that directs STAT-regulated expression of the puromycin resistance gene.
• Hygromycin-resistant cells were subsequently infected with CIS"prey"-expressing retrovirus for 24-48 hours. Retroviral gene transfer was chosen to attain expression from single integrants (Kitamura et al., 1995; Kojima and Kitamura, 1999).
• Cells were treated with Epo (50 ng/ml) for another 24-48 hours prior to puromycin selection. As shown in Figure 9C, colony formation was only observed in Epo-stimulated HEK293-16 cells co- expressing EpoR-"bait" and gp130-CIS"prey” proteins. FACS analysis with anti-FLAG antibody of permeabilized, puromycin resistant cells confirmed expression of the "prey" polypeptide ( Figure 9D).
• Figure 9E shows the results of a "spiking" experiment where a complex retroviral HEK293 cDNA library was mixed with a dilution series of retrovirus expressing the gp130-CIS "prey". A dose-dependent recovery of cell clones was observed, only in the presence of ligand. RT-PCR cycle sequencing in a parallel experiment allowed identification of gp130-CIS"prey" expression in 19 out of 21 clones analysed.
• Example 6 Library screening with the MAPPIT system A screening experiment with the EpoR"bait" using a retroviral HEK293 cDNA library (2.10 6 independent clones) was performed. To favour single integrants, 6.10 7 HEK293- 16 cells expressing the EpoR"bait" were infected with an estimated infection efficiency of 4 %. Three weeks after Epo stimulation and selection in medium containing 2 ⁇ g/ml puromycin, 33 colonies were picked and analysed in a functional assay (Figure 9F).
• Example 7 the use of MAPPIT with heterodimeric receptors: Functionality of IL3R-, IL5R- and GM-CSFR-LepR chimeras in the Hek 293T cell line
• Functionality of IL3R-, IL5R- and GM-CSFR-LepR chimeras in the Hek 293T cell line In order to compare the functionality of the Epo, IL-3R, IL-5R and GM-CSFR LepR- chimera, following combinations of plasmids were transfected into 4x10 5 Hek293T cells, seeded the day before transfection: a. pSV-SPORT-EpoR/LepR + pGL3-rPAP1-luci + pUT651 b.
• EpoR/LepR chimera and a combination of IL-3R ⁇ /LepR with ⁇ c /LepR have similar fold inductions.
• a signal above background is observed at cytokine concentrations of 1 ng/ml.
• the biological activity of IL-5 on cells transfected with the chimera IL-5R ⁇ /LepR and ⁇ c/LepR is less than these for IL-3 or Epo.
• Cells transfected with chimeras of the GM-CSFR and the LepR are much more sensitive to stimulation, with a clear 7.7 fold induction at a concentration as low as 10 pg/ml.
• pSV-SPORT- ⁇ c /LepR-F3-Smad3 + pSV-SPORT-GM-CSFR ⁇ /LepR-F3-ALK4CA + pMG2 + pXP2d2-rPAP1-luci + pUT651.
• a precipitation mixture of 300 ⁇ l was prepared which contained 2.92 ⁇ g (0.02 ⁇ g for pUT651, 0.2 ⁇ g for pXP2d2-rPAP1-luci + 0.9 ⁇ g of each of the others). 200 ⁇ l of this mixture was applied to the cells. After 6 hours, the cells were washed once with Dulbecco's PBS, and DMEM medium was added.
• the cells were resuspended using 200 ⁇ l Cell Dissociation Agent. After neutralization with 2200 ⁇ l DMEM medium, 40 ⁇ l of this cell suspension was brought into a 96 well plate (Costar). The cells were stimulated in a final volume of 100 ⁇ l with final concentrations of 1 ng/ml GM-CSF, 1 ng/ml GM-CSF plus 10 ⁇ M forskolin, 10 ⁇ M forskolin or were left unstimulated. 24 hours after stimulation, luciferase and galactosidase activity assays were performed as described above.
• Example 9 the use op MAPPIT to screen compound-compound interactions comprising non-polypeptide compounds
• FK506 is chemically linked to cyclosporin A (e.g. WO 94/18317).
• bivalent compounds are obtained by fusing FK506 to tetracycline or to a steroid ligand.
• Such hybrid compounds have the capacity to interact with both protein partners: FKBP12 and cyclophilin (or tetracycline/steroid receptors).
• SR alph promoter an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 log terminal repeat. Mol. Cell. Biol., 8, 466 - 472.

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